Many attempts to modify HIV-1 Envelope (Env) for improved immunogenicity have been attempted, with varying rates of success. Some increased potency in neutralizing antibodies (NAbs) has been claimed, but the breadth achieved is very limited and a breakthrough is crucial to make substantial progress towards an effective Env-based vaccine component. Superinfection (SI) provides a unique immunological setting for studying candidate HIV Envs as potential vaccine components. This project focuses on Env sequences derived from superinfected individuals identified to have characteristics of elite neutralization breadth. The Env sequences from these individuals may contain unique determinants that have developed as the result of the persistence of dual antigen presentation during SI. The project objective is to conduct immunogenicity studies using carefully selected panels of vaccine candidates combined with newly emerging vaccine regimens that will result in substantially improved antibody responses - both in time to response and in breadth of neutralization. To achieve the goal of this project, Single Genome Amplification will be used to clone a panel of gp160 Env genes isolated from plasma and targeting time points associated with development of potent neutralization breadth from two intersubtype superinfected elite neutralizers who are members of a well-characterized African sex-worker cohort. A set of selection criteria has defined key time points during SI from which to clone Envs that are expected to have the greatest probability of containing critical determinants that led to a peak in neutralization breadth. In silico analysis will be carried out to assess the evolution of the diversity and divergence of the cloned Envs from each subject by generating a maximum-likelihood phylogenetic tree. Mutations post SI will be mapped, including recombination events between the two infecting viral strains. Potential N-linked glycosylation sites will be examined and other mutations that are known to cause resistance to several broadly neutralizing monoclonal antibodies. Effects of directional selection pressure (dN/dS) will also be evaluated. Following a full in silico analysis of all clones, vaccine candidates will be selected and made as functional pseudoviruses to test for autologous and heterologous neutralization sensitivity. Nucleotide sequences of selected vaccine candidates will be motif optimized and synthesized in preparation for full-length gp160 constructs. Gene Gun bullets will be prepared and site-directed mutagenesis will generate soluble gp140 trimeric protein for expression and purification. Comparative immunogenicity studies will be performed in rabbits using gp160 DNA combined with gp140 trimeric protein to determine which combinations of Envs derived from SI subjects are most effective in generating neutralization breadth. In the process, we expect to define novel, key epitopes that direct the development of neutralizing antibodies. This study will result in a new collection of Env-based immunogens with the potential to advance vaccine research.